| Product code | Prefix (designates the instrument): SCB, SCBS, SPP, SCBI, SPSM, SCH, SPMX, SCR, SCS, SD + Add: PAP, PAD50L, PAD200L, PAHC30M, PAHC200M Example: SCBS PAHC200M |
|---|---|
| Intended purpose | Site-directed, reversible capture immobilization of antibodies and Fc fusion proteins under physiological conditions. Recommended applications include the investigation of biomolecular kinetics involving proteins, peptides, nucleic acids and small molecules. |
| Storage | Store at -20 °C, desiccated over molecular sieve 4A or at 2–8 °C in physiological buffer. |
| Related products |
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PAP, PAD, and PAHC sensor chips are coated with a bioinert polycarboxylate matrix, pre-functionalized with recombinant 47 kDa Protein A. This non-glycosylated Protein A variant includes five IgG-binding domains aligned in series, enabling high-affinity binding to the Fc region of select mammalian antibodies, especially human IgG1, IgG2, and IgG4, as well as Fc-tagged biomolecules under physiological conditions. Quantitative regeneration of the binding complex is efficiently achieved with brief pulses of glycine·HCl at pH 1.5, or in alkaline conditions as needed.
XanTec’s Protein A sensor chips are ready-to-use, eliminating the need for time-intensive assay optimization and streamlining workflows. With five distinct Protein A-modified versions available for SPR applications, each is tailored to specific analytical needs:
Together, these features make XanTec Protein A sensor chips a versatile and reliable choice for SPR applications, whether for kinetic studies or antibody quantification.
Protein A-binding ligand (to be provided by the user)
Glycine buffer pH 1.5 (product code B G15-50ML): 10 mM Glycine·HCl pH 1.5
Optional: Conditioning buffer 1 (product code B C1-50ML): 50 mM NaOH, 1 M NaCl
Ensure that the flow system of your SPR equipment is free from any protein contamination, as even small amounts of desorbed protein can accumulate on the charged sensor surface. If necessary, clean the system using either 1 % Tween 20 or, for a more stringent cleaning, 0.5 % SDS for 5 minutes, followed by 50 mM glycine·HCl (pH 9.5) for 10 minutes (both included in the Desorb Kit, product code K D-500ML). The glycine is required to remove residual traces of SDS.
Allow the sealed sensor chip pouch to equilibrate at room temperature to prevent condensation on the chip surface.
After opening the pouch, install the sensor chip by following the instrument manufacturer’s instructions.
Note: XanTec SPR sensor chips, like all nanocoatings, are prone to degradation when exposed to the atmosphere due to reactive oxygen species in the air. To prevent this, unmounted sensor chips should be stored in a closed container under an inert gas atmosphere or in a physiological buffer for short-term storage.
| Procedure | Flowrate [µL/min] |
Injection time [s] |
|
|---|---|---|---|
| 1 | Equilibrate your SPR-system with physiological running buffer like HBSTE or HBSTE+ and mount a compatible XanTec Protein A sensor chip. | ||
| 2 | Condition all channels with glycine·HCl pH 1.5. Wait until the baseline has stabilized. |
25 | 5x60 |
| 3 | Divert your reference channel and inject your protein A-binding antibody at concentrations of 1–20 µg/mL. Low ligand concentrations and flow rates are recommended to achieve good control over ligand immobilization levels. Adjust conditions and repeat ligand injection as required to achieve the target immobilization level. Wait until the baseline has stabilized. |
1–10 | 60–300 |
| 4 | Start interaction analysis. | ||
| 5 | Regenerate the surface with glycine·HCl pH 1.5. Repeat as required to achieve quantitative removal of antibody-antigen binding complex | 25 | 30 |
Avoid prolonged incubation of the sensor chip in water, as this can negatively affect the integrity of the sensor coating over time. Instead, use a physiological buffer like HBSTE.
If regeneration with glycine·HCl pH 1.5 is insufficient, inject short 30 s pulses of Conditioning buffer 1. Please note that alkaline regeneration conditions should be avoided whenever possible to prevent baseline drift and minor initial capacity loss.
For later reuse, sensor chips can be stored either dry or wet under physiological conditions. When handling the sensor chip, avoid touching the top coating with gloves or tweezers. Reuse of planar PAP sensor chips is not recommended due to the lower overall stability and robustness of planar sensor coatings.
Biacore users only: To prevent detachment of the glass chip in the instrument after chips have been stored under buffer or at 100 % humidity, we strongly recommend checking the mechanical stability of the assembly before inserting the chip cartridge into the instrument.
Reichert users only: If the sensor chip is intended for later reuse, use the refractive index matching foil instead of immersion oil when installing the sensor chip for the first time. Oil traces may contaminate the hydrogel top coating after chip removal, potentially causing irreversible damage to the immobilized protein A.
| Dry storage | |
|---|---|
| 1 | Dismount the used sensor chip from your SPR instrument. |
| 2 | Rinse the hydrogel surface of the sensor chip carefully with ultrapure water. |
| 3 | Dry the sensor chip with a jet of filtered air or nitrogen. |
| 4 | Store the sensor chip dry, using a 3A or 4A molecular sieve, in a cold environment (-25 °C) under an inert gas atmosphere in a tightly sealed container. The underlying hydrogel coating as well as the pre-immobilized protein A should remain stable for several weeks to months. |
| 5 | Store the sensor chip dry, using a 3A or 4A molecular sieve, in a cold environment (-25 °C) under an inert gas atmosphere in a tightly sealed container. The stability of the sensor chip depends on the stability of the immobilized ligand. The underlying hydrogel coating should remain stable for several weeks to months. |
| 6 | Reinstallation Equilibrate the sensor chip to room temperature before opening the storage container, then insert the chip according to the instrument manufacturer‘s instructions. |
| Wet storage | |
|---|---|
| 1 | Dismount the used sensor chip from your SPR instrument. |
| 2 | Rinse the chip surface carefully with ultra-pure water. Place the sensor chip in a container filled with sterile filtered, physiological buffer and seal it tightly. For Cytiva sensor chips, 50 mL centrifugation tubes are applicable. Store the sensor chip refrigerated at 2–8 °C. |
| 3 | Reinstallation Remove the sensor chip from the container, preferably using clean tweezers. Rinse with ultra-pure water to remove buffer salts, and carefully dry it using a jet of filtered air or nitrogen. Then, insert the chip according to the instrument manufacturer‘s instructions. |
| Issue | Possible solution |
|---|---|
| Inefficient regeneration | In general, antibody regeneration under acidic conditions is effective. Use repeated 30-second injection pulses of glycine·HCl (pH 1.5). Alternatively, apply 30-second pulses of alkaline Conditioning Buffer 1; however, note that this step may cause some baseline drift. Inject 4 M MgCl₂ to further optimize regeneration. |
| Instable baseline | If signal drift persists after ligand immobilization, try reducing the antibody capture level to stabilize the baseline. If lower capture levels result in insufficient analyte binding signals, consider switching to a sensor chip with higher capacity, such as PAD200L or PAHC200M. Some IgG subclasses may not bind to the Protein A sensor chip with sufficient affinity. In such cases, switching to another capture system with high-affinity secondary antibodies is recommended. |
| High Nonspecific binding | Increase the Tween concentration in your running buffer (e.g., switch from HBSTE to HBSTE+). Add BSA to your running buffer at concentrations of 0.02–3 %. |
V. 11/24a
For in-vitro use only. Not for use in clinical diagnostic procedures.